To study the bioactive polypeptides included in Bufo skin and its secretions the plasmid skin cDNA library of adult Japanese toad Bufo japonicus formosus was prepared. The pSD64TR has been used as the vector and the cloning sites are Xho I and EcoR I. To screen cDNAs encoding bioactive components, the plasmid cDNA library was transformed into E. coli DH5 competent cells, and positive colonies were screened by colony PCR (polymerase chain reaction). The suspension of a single colony in LB medium was used as the template, SP6 (the upstream primer of the plasmid cDNA library) and a primer with Xho I site and polyT were used as the primers. As the result, 465 positive colonies out of 1 344 were obtained and their plasmid were collected and sequenced. By homologous analysis, it was found that one of the cDNAs encoding a peptide with high homolog with transgelin-2, which was registered in GenBank (accession number: JX197456), and it was indicated as a partial cDNA sequence with a deletion at the 5' end. The transcript is 997 bp consisting of 31 bp 5', 618 bp 3' untranslated region (UTR) and an open reading frame (ORF) of 348 bp encoding a polypeptide of 115 amino acids. In the putative protein product, there is a calponin homology domain, two cysteine residues for a disulfide bond and three a-helix domains, and five potential phosphorylation sites. The homologous analysis indicates 90% similarity with Xenopus (Silurana) tropicalis and 89% with Xenopus laevis, and 71%-85% with other species.
Amino Acid Sequence ; Animals ; Base Sequence ; Bufonidae ; genetics ; metabolism ; Cloning, Molecular ; Gene Library ; Microfilament Proteins ; chemistry ; genetics ; metabolism ; Muscle Proteins ; chemistry ; genetics ; metabolism ; Open Reading Frames ; Phosphorylation ; Phylogeny ; Plasmids ; genetics ; Sequence Homology, Amino Acid ; Skin ; metabolism ; Xenopus ; genetics

OBJECTIVETo isolate and identify TRF1 immunoprecipitating protein complex and to clone the candidate gene.

METHODSThe co-immunoprecipitation assay was employed to isolate TRF1 protein complex and the immunoprecipitate was subjected to MALDI-TOF mass spectrometry for protein identification. The candidate gene was amplified by temperature-gradient PCR from human testis cDNA library and then cloned into pEGFP-C2 vector for eukaryotic expression. The amplified gene was verified by direct sequencing and GFP-tagged protein was confirmed by immunoblotting.

RESULTSTara protein with the size of 68 kD was identified from the TRF1 precipitate. The candidate gene amplified from cDNA library was about 1.7 kb as expected. Sequencing demonstrated the amplified fragment had 99.9% of homogenesis with Tara CDS sequence (gi:30474869). GFP-tagged fusion protein was about 100 kD. Tara was diffusely distributed in cytoplasm at interphase and in whole cells at mitotic phase.

CONCLUSIONTara might be an interacting protein with TRF1. However, further investigation would be required to confirm if they were bona fide partners.

Cloning, Molecular ; HeLa Cells ; Humans ; Microfilament Proteins ; genetics ; isolation & purification ; metabolism ; Protein Binding ; Telomeric Repeat Binding Protein 1 ; chemistry ; genetics ; metabolism

Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Acanthamoeba healyi, a causative agent of granulomatous amebic encephalitis. This clone exhibited high similarity with genes of Physarum polycephalum and Dictyostelium discoideum, which encode actin bundling proteins. Domain search analysis revealed the presence of essential conserved regions, i.e., an active actin binding site and 2 putative calcium binding EF-hands. Transfected amoeba cells demonstrated that AhABP is primarily localized in phagocytic cups, peripheral edges, pseudopods, and in cortical cytoplasm where actins are most abundant. Moreover, AhABP after the deletion of essential regions formed ellipsoidal inclusions within transfected cells. High-speed co-sedimentation assays revealed that AhABP directly interacted with actin in the presence of up to 10 micrometer of calcium. Under the electron microscope, thick parallel bundles were formed by full length AhABP, in contrast to the thin actin bundles formed by constructs with deletion sites. In the light of these results, we conclude that AhABP is a novel actin bundling protein that is importantly associated with actin filaments in the cytoplasm.
Transfection ; Sequence Analysis, DNA ; Sequence Alignment ; Microscopy, Electron, Transmission ; Microfilament Proteins/*chemistry/genetics/*metabolism ; EF Hand Motifs ; DNA, Complementary ; Culture Media ; Cloning, Molecular ; Animals ; Amino Acid Sequence ; Actins/*metabolism ; Acanthamoeba/genetics/growth & development/*metabolism